and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in three differentially TCDD-sensitive rat lines.

Transcription

1 ToxSci Advance Access published April 14, 2004 Pattern of male reproductive system effects after in utero and lactational 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure in three differentially TCDD-sensitive rat lines. Ulla Simanainen 1,2, Tapio Haavisto 3, Jouni T. Tuomisto 1, Jorma Paranko 3, Jorma Toppari 4, Jouko Tuomisto 1,2, Richard E. Peterson 5, and Matti Viluksela 1 1 National Public Health Institute, Department of Environmental Health, P.O. Box 95, FIN Kuopio, Finland 2 University of Kuopio, P.O. Box 1687, FIN Kuopio, Finland 3 Department of Biology, Laboratory of Animal Physiology, University of Turku, FIN Turku, Finland 4 Departments of Physiology and Pediatrics, University of Turku, FIN Turku, Finland 5 School of Pharmacy and Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin 53705, USA. Running title: Perinatal TCDD effects on different rat lines Address of correspondence: Ulla Simanainen P.O. Box 95 FIN Kuopio, FINLAND telephone: fax: ulla.simanainen@ktl.fi Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 1/27 Toxicological Sciences Society of Toxicology 2004; all rights reserved.

2 Pattern of male reproductive system effects after in utero and lactational 2,3,7,8- tetrachlorodicbenzo-p-dioxin (TCDD) exposure in three differentially TCDD-sensitive rat lines. Ulla Simanainen, Tapio Haavisto, Jouni T. Tuomisto, Jorma Paranko, Jorma Toppari, Jouko Tuomisto, Richard E. Peterson, and Matti Viluksela. Abstract Male reproductive effects induced by in utero and lactational exposure to TCDD were analyzed in three differently TCDD sensitive rat lines. Line A, B and C rats are selectively bred from TCDD-resistant Han/Wistar [Kuopio] (H/W) and TCDD-sensitive Long-Evans [Turku/AB] (L-E) rats and exhibit highly different LD50 values for TCDD: >10000, 830, and 40 µg/kg in males, respectively. The resistance in line A rats is linked to a mutated H/W-type aryl hydrocarbon receptor (Ahr hw ) and in line B rats to a H/W-type unknown allele B (B hw ). Line C rats have no resistance alleles. Influence of the resistance alleles on developmentally induced male reproductive effects of TCDD was studied by exposing pregnant females to TCDD (0.03, 0.1, 0.3, or 1 µg/kg) on gestation day (GD) 15. Male progeny were sacrificed on postnatal day (PND) 70. Secondly, the dams were given 1 µg/kg TCDD on GD15 and male progeny sacrificed on PND 14, 21, 28, 35, or 49. Serum testosterone concentration, male sex organ weights, as well as testicular and cauda epididymal sperm numbers were analyzed. Decreased sperm numbers was the most sensitive endpoint. The dose of 1 µg/kg TCDD reduced daily sperm production by 9.3, 25, and 36%, and cauda epididymal sperm reserves by 18, 42, and 49% in line A, B and C rats when measured on PND 70, respectively. The most consistent and significant effects were decreased weight of prostate lobes. The growth of the male reproductive organs was not markedly affected by the resistance alleles Ahr hw and B hw. In contrast, the effects on sperm parameters appear to be slightly modified by the resistance alleles. Thus the intraspecies genetic differences in C-terminal transactivation Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 2/27

3 domain of AHR appear to modify the sensitivity to only certain dioxin-induced male reproductive effects. Key words: 2,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD, dioxin, in utero and lactational exposure, male reproductive tract, strain differences, rat, prostate, sperm Introduction The endocrine system presents a number of target sites for the induction of adverse effects by the ubiquitous environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Numerous studies have demonstrated that male reproductive and developmental processes are sensitive to perinatal low-dose TCDD exposure, resulting in e.g. reduced sperm number and reduced size of reproductive organs (reviewed by Peterson et al. 1993; Theobald et al. 2003). These low-dose effects are essential in terms of risk assessment of TCDD and similar types of compounds. Sharpe and Skakkebaek (1993) hypothesized that in utero exposure to environmental endocrine disrupters such as TCDD may, at least in part, be responsible for decreased human sperm counts and other male reproductive tract disorders. However, epidemiological evidence is not yet available to support this hypothesis. Various dose-dependent effects on male offspring androgenic status and growth of androgendependent organs have been reported in different strains of pregnant female rats administered with TCDD (Faqi et al. 1998; Gray et al. 1997a; Haavisto et al., 2001; Mably et al. 1992a,b,c; Ohsako et al. 2001, 2002; Wilker et al. 1996). The range of defects and the TCDD sensitivity has varied between the studies, even within the same rat strain. Mably et al. (1992c) and Faqi et al. (1998) reported that daily sperm production was highly susceptible to perinatal TCDD exposure in Holtzman and Wistar rats. However, the effect was not reproducible in Sprague-Dawley and Long-Evans rats (Gray et al. 1997a; Wilker et al. 1996), Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 3/27

4 or in another study with Holtzman rats (Ohsako et al. 2001). Decreased sex accessory organ weights were observed in most studies, though the magnitude of the defects varied among studies. The most sensitive, persistent and reproducible effects were seen on sperm numbers, especially the decrease in cauda epididymal, and ejaculated sperm numbers. In our laboratory, two rat strains with a great sensitivity difference to TCDD-induced acute lethality have been used (Pohjanvirta and Tuomisto, 1994). In Han/Wistar (Kuopio) (H/W) rats, the LD 50 for TCDD is over 10,000 µg/kg whereas for Long-Evans (Turku/AB) (L-E) rat it is about 10 µg/kg. The TCDD resistance of H/W rats is due to a mutated Ahr allele (Ahr hw ) and to the currently unknown allele B hw (Pohjanvirta et al. 1998; Tuomisto et al. 1999). The Ahr hw allele has been shown to harbor a point mutation that results in an abnormal C- terminus transactivation domain and a smaller AHR protein in H/W than in L-E rats (~98 kda vs. 106 kda). L-E rats have clearly higher total hepatic levels of AHR than H/W rats (Pohjanvirta et al. 1998, 1999; Franc et al. 2001). However, no difference between the strains were found in TCDD affinity to cytosolic AHR, in ability of their AHRs to be transformed into the DNA-binding form by TCDD, or in the specific binding of the activated AHR to DNA (Pohjanvirta et al. 1999). Also the up-regulation of the AHR by TCDD was similar in H/W and L-E rats (Franc et al. 2001). The identity of B allele has not yet been determined, but it may encode a protein participating in the AHR signalling pathway. However, it was recently shown that at least the differences in relative expression of AHR dimerization partner ARNT or the AHR repressor (AHRR), do not contribute to the dioxin sensitivity differences between the H/W and L-E rats (Korkalainen et al. 2003, 2004). Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 4/27

5 Recently, two H/W-type TCDD resistance genes (Pohjanvirta 1990), the altered Ahr hw and another unknown allele B hw were segregated into two new rat lines designated A and B (Tuomisto et al. 1999). Line A has the resistance allele Ahr hw and line B has the resistance allele B hw. Line C has only wild-type alleles Ahr wt /B wt. Lines A, B, and C exhibit highly different LD 50 values for TCDD: >10,000, 830, and 40 µg/kg in males, respectively. The typical endpoints of dioxin toxicity can be classified based on the modification of TCDD effects by the resistance alleles Ahr hw and B hw (Simanainen et al. 2002; 2003). The type I endpoints (increased EROD activity, decreased thymus weight, tooth defect) are independent of genotype variation. However, for type II endpoints (e.g. weight loss, liver toxicity, increased serum bilirubin), the efficacy (magnitude of effect) of TCDD is suppressed by the resistance alleles. The Ahr hw allele apparently is the most important resistance factor. In contrast to other rat strains studied (Haavisto et al. 2001; Mably et al. 1992a; Roman et al. 1995), maternal TCDD exposure on gestational day (GD) 13 stimulated testicular testosterone synthesis and increased circulating testosterone concentrations in H/W fetuses (GD 19.5) (Haavisto et al. 2001). Therefore, it was hypothesized that the mutated H/W-type AHR could modify the in utero TCDD effects on male reproductive endpoints. The majority of single nucleotide polymorphisms found in human Ahr locate in exon 10, which covers the major portion of the C-terminal transactivation domain (Cauchi et al. 2001; Harper et al. 2002; Kawajiri et al. 1995; Smart and Daly 2000; Wong et al. 2001). Interestingly, a recent study showed that a combination of two or three of single nucleotide polymorphisms (SNPs) found in the C-terminal end of the human Ahr failed to induce Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 5/27

6 TCDD-dependent CYP1A1 expression in vitro (Wong et al. 2001). Therefore, the sensitivity differences based on the mutated Ahr could have relevance for human risk assessment, although there is currently no evidence for the clinical relevance of the Ahr polymorphism in humans. The aim of the present study was to determine whether the sensitivity difference to TCDD acute lethality among the rat lines A, B and C correlates with the magnitude of the developmental male rat reproductive tract changes induced by perinatal TCDD exposure. Accordingly, the dose-response and time-course of in utero and lactational TCDD exposure effects on male reproductive organ weights, serum testosterone concentrations, cauda epididymal sperm number, and daily sperm production were determined in rat lines A, B, and C. Materials and methods Dose-response study. Line A, line B and line C rats were bred in the SPF (specific pathogen free) barrier unit of the National Public Health Institute (Kuopio, Finland). Adult (12-15 week old) female rats in estrus were mated with untreated males from 9 to 12 AM and vaginal smears were collected and examined for the presence of sperm. The day after copulation was considered gestation day (GD) 0. Pregnant females were housed singly in plastic cages that had wire-mesh covers in a room with a 12 hours light: dark cycle (lighted from to 19.00). Aspen chips (Tapvei Co., Kaavi, Finland) were used as bedding and nesting material. The temperature in the animal Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 6/27

7 room was 21 ± 1 C and the relative humidity 50 ± 10%. Rats were maintained on standard pelleted laboratory animal feed (R36, Ewos, Södertälje, Sweden) and tap water ad libitum. In the morning on GD 15, graded single doses of TCDD (0.03, 0.1, 0.3 or 1 µg/kg) or an equivalent volume of vehicle (corn oil), 4 ml/kg, were given by gavage to pregnant rats. For each dose, 5-8 pregnant dams were used. TCDD was purchased from the UFA-Oil Institute (Ufa, Russia) and it was over 99% pure as confirmed by gas chromatography-mass spectrometry. The day of birth was considered postnatal day (PND) 0. One day after birth, the number of live offspring and the sex ratio were recorded. When possible, by random termination of excess offspring, litters were adjusted to 3 males and 3 females to allow uniform postnatal exposure. In the rat lines used, the average number of pups per litter is only 6 ± 3 (mean ± SD). In order to keep the litters as homogenous as possible and without greatly increasing the number of pregnant dams needed, only litters with less than four pups were excluded. Offspring were weaned on PND 28 terminating the lactational TCDD exposure. Dams were killed and the number of uterine implantation sites was counted after staining the uteri with ammonium sulfide solution (10%, v/v). For each litter, survival from implantation to the day after birth was calculated by the following formula: 100% b/a [1] (a = total number of implantation sites, b = number of pups on PND 1). After weaning, the pups were housed with the same-sex littermates in plastic cages with wire-mesh covers. Maternal and pup viability was monitored throughout the study. Body weight of the offspring was determined on PND 1, 4, 7, 14, and 28. Anogenital distance (AGD) and crown-rump Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 7/27

8 length (CRL) were measured on PND 1 and PND 4 by using a vernier caliber capable of resolution to 0.1 mm. At the age of 70 days, the pups were decapitated and trunk blood collected. For testosterone analysis, sera were stored at 80 C. Testes, cauda of the right epididymis, ventral prostate, seminal vesicles and thymus were dissected and weighed. Seminal vesicles were weighed without fluid and coagulating glands. The organ weights were reported as both absolute and relative weights (organ weight/body weight). Cauda epididymis and testes were frozen for the analysis of sperm counts. Time-course experiment. Pregnant rats were dosed by gavage with a single dose of 1 µg/kg TCDD or an equivalent volume of corn oil vehicle (4 ml/kg) on GD15. One or two male pups from each litter were terminated at PND 14, 21, 28, 35, or 49. There were 4-8 pregnant dams per group and 7 ± 2 (mean ± SD) male pups were examined at each time point. On PND1, the number of live offspring and sex ratio were recorded and the offspring examined for gross malformations. When possible, litters were adjusted to 4 males and 2 females. Body weights of offspring were determined weekly from PND 1 until PND 49. At termination, the male pups were decapitated, and trunk blood was collected. Separated sera were stored at 80 C. Testes, right cauda epididymis, ventral prostate, dorsolateral prostate, anterior prostate and seminal vesicles were dissected and weighed. Fluid was not removed from the seminal vesicles. Right cauda epididymis and testis were frozen for sperm Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 8/27

9 count analysis. Additionally, for male pups terminated on PND 35 the lungs, heart, kidneys, liver, salivary glands and spleen were dissected and weighed. Serum testosterone. Serum testosterone concentrations were analyzed from all samples in the time-course experiment and from the control and highest TCDD dose (1 µg/kg) samples in the doseresponse study. Testosterone was measured from diethyl ether extracted sera by timeresolved fluoroimmunoassay, DELFIA (Perkin Elmer Life and Analytical Sciences, Wallac Oy, Turku, Finland). After extraction, samples were reconstituted to 100 µl of Dilution II buffer (Perkin Elmer Life and Analytical Sciences, Wallac Oy) from which 25 µl/well was taken for analysis. For enhancing the sensitivity of the assay, commercial tracer and antisera were additionally diluted 5:8 giving a sensitivity of 0.04 ng/ml when the limit of detection was defined as the value two standard deviations above the mean of the zero standard measurement value. Intra- and inter-assay variations were below 6 and 12%, respectively at testosterone concentration of 1.0 ng/ml. Daily sperm production and cauda epididymal sperm counts. For sperm counts, frozen testis and cauda epididymis were homogenized for 2 min using Ultra Turrax homogenizer (model T25 basic, IKA-WERKE GMBH & CO, Germany) in 50 ml (testis) or 20 ml (cauda epididymis) 0.9 % saline, containing 0.05% Triton X-100 and 0.01% thimerosal. Homogenates were diluted to approximately 1 x 10 6 sperm/ml, and counts from 4 hemocytometer chambers were counted and averaged (Blazak et al. 1993). Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 9/27

10 Statistical analysis. Statistical analysis was performed using SPSS 10.0 Statistical program (SPSS Inc., Chicago, IL, USA). Litter means were used and data were analyzed by one-way analysis of variance (ANOVA). If the test showed a significant difference, the least significant difference test was used as a post hoc test. In case of nonhomogenous variances (according to Bartlett s test, p < 0.01), the non-parametric Kruskal-Wallis ANOVA was used followed by the Mann-Whitney U test. P values less than 0.05 were considered significant. Results Maternal toxicity and pup survival. TCDD did not produce any deaths or overt toxicity to the dams as assessed by body weight gain and visual inspection (data not shown). In addition, pup survival from implantation to the day after birth was not affected by TCDD. However, survival from implantation to the day after birth was surprisingly low in control line B rats in the dose-response study (41 %) resulting in a significant difference compared with the two lowest doses (0.03 and 0.1 µg/kg TCDD). The average survival was 85 % (range 80-89%), 64% (41-86%) and 74% (63-85%) in control line A, B and C rats, respectively. Corresponding values after TCDD exposure were (without clear dose-response) 83% (70-92%), 60% (28-85%), and 77% (61-86%), respectively. For each line the percentage of male pups survived between PND1 and weaning on PND28 was 99 %. The only exceptions were line B males exposed to 0.03 µg/kg TCDD and line C males exposed to 0.03 or 1 µg/kg TCDD, where the male survival rate was in average 81% (range 81 83%). Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 10/27

11 Non-reproductive organ weights on PND35. The relative weight of liver, spleen, heart, lung and kidneys tended to be increased in line B and C male offspring. However, the increase was significant only for relative liver weight in line A and C males and for relative lung weight in line C males (Table 1). Growth of the male offspring. The dose-response study showed that on PND 70 significant reduction in body weight was observed only in line B and C rats at the highest dose of 1 µg/kg TCDD (Table 2). However, at younger ages in the time-course study, the same dose significantly reduced body growth in all lines and at most time points measured (Table 2). Exceptionally, in 1-day-old line A males exposed to 0.03 µg/kg TCDD the body weight was increased compared to control (data not shown). Anogenital distance. Anogenital distance (AGD) was measured on PND 1 and 4 (Table 3). On PND 1, the highest maternal dose of 1 µg/kg TCDD significantly decreased the absolute AGD only in line C rats. On PND 4, the AGD did not differ from controls in any group. In addition, when AGD was related to the cube root of pup body weight [AGD/(body weight) 1/3 ] (Gallavan et al. 1999), there was no detectable effect between the TCDD-exposed and control rats at either time-point. Serum testosterone. Serum testosterone levels in line A, B and C male rats were low, below 1 ng/ml and were not significantly affected by TCDD (data not shown). Pubertal increase in testosterone levels of line A and B control animals was detected on day 49. However, in line C male rats, serum testosterone levels in the control animals and those exposed to TCDD remained low until day 49, but the increase was present on day 70 (testosterone measured only in the 1 µg/kg TCDD dosing group). Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 11/27

12 Accessory sex organ weights. Until day 49, the maternal dose of 1 µg/kg TCDD decreased both absolute and relative weight of the ventral, anterior and dorsolateral prostate in line A, B and C rats at most time points measured. The change was most consistent and significant in the ventral lobe (Table 2). In the group exposed to 1 µg/kg TCDD, the average decrease in absolute weight of anterior prostate was 37% (range 30-55%), 32% (18-42%) and 34% (19-45%) in line A, B, and C rats, respectively. Similarly the average dorsolateral prostate weight was decreased by 34% (range 29-37%), 28% (16-33%) and 39% (22-59%), in line A, B, and C rats, respectively. The effect on ventral prostate was not permanent, as the only significant decrease in weight remaining at the age of 70 days was observed in line B rats at maternal dose of 1 µg/kg TCDD (Table 3). TCDD did not have any consistent effects on the weight of seminal vesicles (data not shown). Testis and epididymis weight. The absolute weights of testis and epididymis are shown in Table 2. Except for a significant increase on PND28-49, the relative weights of testis, epididymis or cauda epididymis remained unchanged. In some individuals a maternal dose of 1 µg/kg TCDD caused severe epididymal malformations including small caput and cauda as well as a degeneration of corpus epididymis. Malformed epididymis was observed in 6 out of 44 line C male rat offspring (6 of 17 litters affected) and 3 out of 47 line A male rat offspring (3 of 15 litters affected). Daily sperm production and cauda epididymal sperm counts. In the dose-response study, the highest TCDD dose (1 µg/kg) reduced daily sperm production by 9, 25, and 36%, and cauda epididymal sperm reserves by 18, 42, and 49% in line A, B and C rats when measured on PND 70, respectively (Fig. 1). In the time-course study the daily sperm production measured Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 12/27

13 in 49-day-old rats had decreased after TCDD exposure by 35% in line A, 17% in line B, and 32% in line C rats. The decrease was significant only in line A rats. Discussion Sensitivity differences among the rat lines. The time-course study indicated that until about 49 days of age the male reproductive system of the three rat lines was similarly affected by in utero and lactational TCDD exposure. However, the dose-response study suggested that compared to line B and C rats, the line A rats were somewhat more resistant to certain endpoints of male reproductive toxicity. At the age of 70 days, sperm parameters (daily sperm production and cauda epididymal sperm reserve) were significantly decreased only in line B and C rats. In addition, the increases in relative weights of the heart, lung and spleen were more pronounced in line C rats than in line A rats. Therefore, it appears that sensitivity of the developing male accessory sex organs to in utero and lactational TCDD exposure is not modified by the resistance alleles Ahr hw and B hw. However, the intraspecies genetic differences in C-terminal transactivation domain of AHR appear to play a role in sensitivity to TCDD-induced decreases in sperm parameters, as well as in the development of certain non-reproductive organs. In a recent study, we were able to show that newborn line A rats are resistant to TCDD acute lethality and that the full resistance develops during the first weeks of postnatal life (Simanainen et al. 2004). In addition, the non-lethal biochemical and toxic short-term effects caused by TCDD exposure in adulthood could be categorized into two types according to the modification by the resistance alleles (Simanainen et al. 2002; 2003; Tuomisto et al. 1999). The efficacy ratio of 0.5 between Ahr hw and Ahr wt genotype was used as a practical classification criterion between type I and type II endpoints (Simanainen et al. 2003). In the Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 13/27

14 present study, the TCDD effect on selected non-reproductive organ weights as well as sperm parameters appeared to fulfill these criteria of type II endpoints with two-fold lower efficacy in line A rats than in line C rats. In contrast, the reduction in prostate weight is a type I endpoint with no sensitivity differences among the rat lines at any age. The deletion mutation of Ahr hw is located within the transactivation domain leaving the domains responsible for ligand and DNA binding as well as heterodimerization intact (Pohjanvirta et al., 1998). Therefore, the activation of transcription seems to be the critical step where the wild-type and H/W-type receptors act differently. This may also be important for the dioxin risk assessment of humans as the majority of polymorphisms in the human AHR is located in the transactivation domain (Harper et al. 2002). In theory, a mutation in human AHR C-terminal end could result in decreased sensitivity to dioxin-induced male reproductive effects as observed in rats with H/W-type mutated AHR. Androgenic status. The lack of effect of TCDD on serum testosterone levels in this study is consistent with previous studies, where a tendency of decrease or no effect (Cooke et al. 1998; Faqi et al. 1998; Gray et al. 1995; Loeffler and Peterson 1999; Roman et al. 1995) were reported in young adults. In the present TCDD-exposed males, testosterone levels were most severely, though not significantly, depressed on day 49. Similar depression was also reported by Roman and coworkers (1995) and is probably associated with the delayed onset of puberty. After 49 days of age, the testosterone levels of TCDD-exposed males approached the control levels. The anogenital distance, which is known to be androgen-sensitive parameter in neonatal rats (Neumann et al. 1970), was not affected by the TCDD exposure. Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 14/27

15 Accessory sex organs. In the present study, no clear effect was seen on relative seminal vesicle weight. Therefore, the seminal vesicle apparently is not as sensitive target as the prostate to in utero and lactational TCDD exposure. Relative organ weights were considered a better indicator than absolute organ weights, because body weight was significantly decreased in each rat line by TCDD and at most time points. The most reproducible effect of in utero and lactational TCDD exposure was the decrease in prostate weight. Prostate weight was decreased at the highest dose, 1 µg/kg TCDD (lower doses were analyzed only on PND70), but the effect was not permanent. Similarly, in Long-Evans hooded (Gray et al. 1997b), Sprague-Dawley (Wilker et al. 1996) and Wistar rats (Faqi et al. 1998), a significant inhibitory effect on prostate development was seen after a single maternal TCDD dose of 0.8, 1 or a total dose of 0.72 µg/kg, respectively. However, in Holtzman rats exposed in utero and via lactation to TCDD, the weight of the ventral prostate and seminal vesicles were significantly reduced after a maternal TCDD dose as low as 0.2 µg/kg until the offspring were 120 days of age (Mably et al. 1992a; Ohsako et al. 2001). These studies imply that although the resistance alleles studied here did not alter the sensitivity of sex accessory organ development to the inhibitory effects of TCDD, there are other strain differences in TCDD sensitivity and persistence of prostate defects with Holtzman rats being the most sensitive. Sperm parameters. Decrease in cauda epididymal sperm counts in particular, is among the most sensitive endpoints of the male rat reproductive pathogenesis after perinatal TCDD exposure (Gray et al. 1995, 1997b; Mably et al. 1992c; Theobald and Peterson, 1997). Inhibitory effects of in utero and lactational TCDD exposure on daily sperm production are less consistent. The present study supports the finding of Ohsako and coworkers (Ohsako et al. 2001) that maternal exposure to a relatively low dose of TCDD (less than 1 µg/kg) has a minimal or no effect on testicular development and spermatogenesis. Similarly, the Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 15/27

16 epididymal sperm reserves were decreased only at the highest maternal doses of TCDD. The effect on sperm parameters was observed on PND 70, which indicate that the effects on sperm numbers are more persistent than those observed in prostate development. Testis and epididymis weight. In accordance with previous studies with Wistar and Holtzman rats (Faqi et al. 1998; Ohsako et al. 2001), the relative weight of testis and epididymis was not affected by in utero and lactational TCDD exposure. There was a slight indication of correlation between the absolute weight of epididymis and cauda epididymal sperm number, and a noticeably weaker correlation or no correlation between absolute weight of testis and daily sperm production. However, the correlation was not strong enough to explain the sensitivity differences between the rat lines. Line C rats remained most susceptible to decreased cauda epididymal sperm number even when the sperm number was related to the epididymis weight. The developmental abnormalities in the corpus epididymis observed were similar to those reported by Wilker et al. (1996) and Ohsako et al. (2002) in Sprague-Dawley rats. Wilker and coworkers (1996) reported partial to complete agenesis of the corpus epididymis in approximately a quarter of the animals after a maternal dose of 2 µg/kg TCDD on GD 15. In the present study, these malformations were observed at 1 µg/kg TCDD and mainly in the sensitive line C rats (14%). This implies that line C rats may be more sensitive to this effect compared to line A and line B rats. In conclusion, despite the great influence of resistance alleles Ahr hw and B hw on TCDD induced acute lethality, the data generated in this study show that these H/W-type alleles (compared with wild-type alleles) do not modify the TCDD effect on male accessory Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 16/27

17 reproductive organ development after in utero and lactational TCDD exposure. However, the alleles may modify TCDD effects on sperm parameters and on the development of some nonreproductive organs, such as thymus, spleen, heart and lung. Differences in developmental sensitivity to TCDD apparently are less profound compared to acute lethality and some other short-term exposure endpoints. The study confirmed that the effects on prostate development and sperm numbers, especially on the epididymal sperm counts, are sensitive and consistent signs of in utero and lactational TCDD exposure. Acknowledgements We are grateful for Ms. Arja Tamminen and Ms. Jaana Jääskö for their excellent technical assistance. This study was financially supported by the Academy of Finland (Grant and Project of the Finnish Research Program on Environmental Health); the European Commission (Contracts QLK4-CT and QLK4-CT ); NIH grant ES01332 (REP); Graduate school in Environmental Health, and Jenny and Antti Wihuri Foundation. References Blazak, W. F., Treinen, K. A., and Juniewicz, P. E. (1993). Application of testicular sperm head counts in the assessment of male reproductive toxicity. In Male Reproductive Toxicology (R. E. Chapin and J. J. Heindel, eds.), Vol. 3A, pp Academic Press, New York, USA. Cauchi, S., Stucker, I., Solas, C., Laurent-Puig, P., Cenee, S., Hemon, D., Jacquet, M., Kremers, P., Beaune, P., and Massaad-Massade, L. (2001). Polymorphisms of human aryl Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 17/27

18 hydrocarbon receptor (AhR) gene in a French population: relationship with CYP1A1 inducibility and lung cancer. Carcinogenesis 22, Cooke, G. M., Price, C. A., and Oko, R. J. (1998). Effects of in utero and lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on serum androgens and steroidogenic enzyme activities in the male rat reproductive tract. J Steroid Biochem Mol Biol 67, Faqi, A. S., Dalsenter, P. R., Merker, H. J., and Chahoud, I. (1998). Reproductive toxicity and tissue concentrations of low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin in male offspring rats exposed throughout pregnancy and lactation. Toxicol Appl Pharmacol 150, Franc, M. A., Pohjanvirta, R., Tuomisto, J., and Okey, A. B. (2001). In vivo up-regulation of aryl hydrocarbon receptor expression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in a dioxin-resistant rat model. Biochem Pharmacol 62, Gray, L. E., Jr., Kelce, W. R., Monosson, E., Ostby, J. S., and Birnbaum, L. S. (1995). Exposure to TCDD during development permanently alters reproductive function in male Long Evans rats and hamsters: reduced ejaculated and epididymal sperm numbers and sex accessory gland weights in offspring with normal androgenic status. Toxicol Appl Pharmacol 131, Gray, L. E., Ostby, J. S., and Kelce, W. R. (1997a). A dose-response analysis of the reproductive effects of a single gestational dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin in male Long Evans hooded rat offspring. Toxicol Appl Pharmacol 146, Gray, L. E., Wolf, C., Mann, P., and Ostby, J. S. (1997b). In utero exposure to low doses of 2,3,7,8-tetrachlorodibenzo-p-dioxin alters reproductive development of female Long Evans hooded rat offspring. Toxicol Appl Pharmacol 146, Haavisto, T., Nurmela, K., Pohjanvirta, R., Huuskonen, H., El-Gehani, F., and Paranko, J. (2001). Prenatal testosterone and luteinizing hormone levels in male rats exposed during Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 18/27

19 pregnancy to 2,3,7,8-tetrachlorodibenzo-p-dioxin and diethylstilbestrol. Mol Cell Endocrinol 178, Harper, P. A., Wong, J. Y., Lam, M. S., and Okey, A. B. (2002). Polymorphisms in the human AH receptor. Chemioterapia 141, Kawajiri, K., Watanabe, J., Eguchi, H., Nakachi, K., Kiyohara, C., and Hayashi, S. (1995). Polymorphisms of human Ah receptor gene are not involved in lung cancer. Pharmacogenetics 5, Korkalainen, M., Pohjanvirta, R., and Tuomisto, J. (2004). Primary structure and inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) of aryl hydrocarbon receptor repressor in a TCDD-sensitive and a TCDD-resistant rat strain. Biochem Biophys Res Commun 315, Korkalainen, M., Tuomisto, J., and Pohjanvirta, R. (2003). Identification of novel splice variants of ARNT and ARNT2 in the rat. Biochem Biophys Res Commun 303, Loeffler, I. K., and Peterson, R. E. (1999). Interactive effects of TCDD and p,p -DDE on male reproductive tract development in in utero and lactationally exposed rats. Toxicol Appl Pharmacol 154, Mably, T. A., Bjerke, D. L., Moore, R. W., Gendron-Fitzpatrick, A., and Peterson, R. E. (1992c). In utero and lactational exposure of male rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin. 3. Effects on spermatogenesis and reproductive capability. Toxicol Appl Pharmacol 114, Mably, T. A., Moore, R. W., Goy, R. W., and Peterson, R. E. (1992b). In utero and lactational exposure of male rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin. 2. Effects on sexual behavior and the regulation of luteinizing hormone secretion in adulthood. Toxicol Appl Pharmacol 114, Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 19/27

20 Mably, T. A., Moore, R. W., and Peterson, R. E. (1992a). In utero and lactational exposure of male rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin. 1. Effects on androgenic status. Toxicol Appl Pharmacol 114, Neumann, F., Berswordt-Wallrabe, R. V., Elger, W., Steinbeck, H., Hahn, J. D., and Kramer, M. (1970). Aspects of androgen-dependent events as studied by antiandrogens. Recent Prog Horm Res 26, Ohsako, S., Miyabara, Y., Nishimura, N., Kurosawa, S., Sakaue, M., Ishimura, R., Sato, M., Takeda, K., Aoki, Y., Sone, H., Tohyama, C., and Yonemoto, J. (2001). Maternal exposure to a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppressed the development of reproductive organs of male rats: dose-dependent increase of mrna levels of 5α-reductase type 2 in contrast to decrease of androgen receptor in the pubertal ventral prostate. Toxicol Sci 60, Ohsako, S., Miyabara, Y., Sakaue, M., Ishimura, R., Kakeyama, M., Izumi, H., Yonemoto, J., and Tohyama, C. (2002). Developmental stage-specific effects of perinatal 2,3,7,8- tetrachlorodibenzo-p-dioxin exposure on reproductive organs of male rat offspring. Toxicol Sci 66, Peterson, R. E., Theobald, H. M., and Kimmel, G. L. (1993). Developmental and reproductive toxicity of dioxins and related compounds: cross-species comparisons. Crit. Rev. Toxicol. 23, Pohjanvirta, R. (1990). TCDD resistance is inherited as an autosomal dominant trait in the rat. Toxicol Lett 50, Pohjanvirta, R., and Tuomisto, J. (1994). Short-term toxicity of 2,3,7,8-tetrachlorodibenzo-pdioxin in laboratory animals: effects, mechanisms, and animal models. Pharmacol Rev 46, Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 20/27

21 Pohjanvirta, R., Viluksela, M., Tuomisto, J. T., Unkila, M., Karasinska, J., Franc, M. A., Holowenko, M., Giannone, J. V., Harper, P. A., Tuomisto, J., and Okey, A. B. (1999). Physicochemical differences in the AH receptors of the most TCDD-susceptible and the most TCDD-resistant rat strains. Toxicol Appl Pharmacol 155, Pohjanvirta, R., Wong, J. M., Li, W., Harper, P. A., Tuomisto, J., and Okey, A. B. (1998). Point mutation in intron sequence causes altered carboxyl-terminal structure in the aryl hydrocarbon receptor of the most 2,3,7,8-tetrachlorodibenzo-p-dioxin-resistant rat strain. Mol Pharmacol 54, Roman, B. L., Sommer, R. J., Shinomiya, K., and Peterson, R. E. (1995). In utero and lactational exposure of the male rat to 2,3,7,8-tetrachlorodibenzo-p-dioxin: impaired prostate growth and development without inhibited androgen production. Toxicol Appl Pharmacol 134, Sharpe, R. M., and Skakkebaek, N. E. (1993). Are oestrogens involved in falling sperm counts and disorders of the male reproductive tract? Lancet 341, Simanainen, U., Tuomisto, J. T., Pohjanvirta, R., Syrjälä, P., Tuomisto, J., and Viluksela, M. (2004). Postnatal development of resistance to short-term high dose toxic effects of 2,3,7,8- tetrachlorodibenzo-p-dioxin (TCDD) in TCDD-resistant and -semiresistant rats. Toxicol Appl Pharmacol., In press. Simanainen, U., Tuomisto, J. T., Tuomisto, J., and Viluksela, M. (2002a). Structure-activity relationships and dose responses of polychlorinated dibenzo-p-dioxins for short-term effects in 2,3,7,8-tetrachlorodibenzo-p-dioxin-resistant and -sensitive rat strains. Toxicol Appl Pharmacol 181, Simanainen, U., Tuomisto, J. T., Tuomisto, J., and Viluksela, M. (2003). Dose-response analysis of short-term effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin in three differentially susceptible rat lines. Toxicol Appl Pharmacol 187, Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 21/27

22 Smart, J., and Daly, A. K. (2000). Variation in induced CYP1A1 levels: relationship to CYP1A1, Ah receptor and GSTM1 polymorphisms. Pharmacogenetics 10, Theobald, H. M., Kimmel, G. L., and Peterson, R. E. (2003). Developmental and reproductive toxicity of dioxins and related compounds. In Dioxins and Health, 2nd edition (A. Schecter and T. A. Gasiewicz, eds.), Chapter 9, pp John Wiley & Sons, New York, USA. Theobald, H. M., and Peterson, R. E. (1997). In utero and lactational exposure to 2,3,7,8- tetrachlorodibenzo-p-dioxin: effects on development of the male and female reproductive system of the mouse. Toxicol Appl Pharmacol 145, Tuomisto, J. T., Viluksela, M., Pohjanvirta, R., and Tuomisto, J. (1999). The AH receptor and a novel gene determine acute toxic responses to TCDD: segregation of the resistant alleles to different rat lines. Toxicol Appl Pharmacol 155, Wilker, C., Johnson, L., and Safe, S. (1996). Effects of developmental exposure to indole-3- carbinol or 2,3,7,8-tetrachlorodibenzo-p-dioxin on reproductive potential of male rat offspring. Toxicol Appl Pharmacol 141, Wong, J. M., Okey, A. B., and Harper, P. A. (2001). Human aryl hydrocarbon receptor polymorphisms that result in loss of CYP1A1 induction. Biochem Biophys Res Commun 288, Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 22/27

23 Tables Table 1: Effects of rat line on absolute organ weights (g) and on the effects of a 1 µg/kg maternal dose of TCDD administered on GD15 on relative organ weights (mg/g body weight) of male offspring at 35 days of age. In average 6 animals per group (range 5-7). Rat line Control (g) Note. Values are means ± SE. Control (mg/g bw) TCDD (mg/g bw) TCDD (% change from control) Kidney weight A 1.20 ± ± ± B 1.13 ± ± ± C 1.02 ± ± ± Thymus weight A 0.25 ± ± ± B 0.36 ± ± ± C 0.35 ± ± ± Spleen weight A 0.46 ± ± ± B 0.53 ± ± ± C 0.41 ± ± ± Heart weight A 0.49 ± ± ± B 0.57 ± ± ± C 0.53 ± ± ± Lung weight A 0.83 ± ± ± B 0.84 ± ± ± C 0.70 ± ± ± 0.24* 25.2* Liver weight A 4.98 ± ± ± 1.43* 9.92* B 5.34 ± ± ± C 4.66 ± ± ± 1.23* 11.4* * Indicates statistically significant (p<0.05) effects of TCDD compared to respective control. Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 23/27

24 Table 2: The effects of gestational day 15 TCDD exposure on body weight (g) and organ weight (mg) in male progeny necropsied at different ages. N = number of litters. PND Rat line Dose (µg/kg TCDD) N Body weight (g) Ventral prostate weight (mg) Testis weight (mg) Epididymis weight (mg) Cauda epididymis weight (mg) 14 A ± ± ± ± ± ± 1.1 * 3 ± 0.2 * 58 ± 1.0 * 7 ± 0.6 * 2 ± 0.1 * B ± ± ± ± ± ± 0.7 * 3 ± 0.3 * 81 ± ± ± 0.1 C ± ± ± ± ± ± 1.7 * 3 ± 0.2 * 64 ± ± ± A ± ± ± ± ± ± 3.0 * 16 ± 1.2 * 150 ± 6.6 * 12 ± ± 0.3 * B ± ± ± ± ± ± 1.4 * 14 ± 1.2 * 190 ± ± ± 0.2 C ± ± ± ± ± ± 2.6 * 10 ± 1.6 * 130 ± ± ± A ± ± ± ± ± ± 1.5 * 32 ± 1.4 * 370 ± 10 * 25 ± ± 0.5 B ± ± ± ± ± ± 1.8 * 24 ± 1.8 * 440 ± ± ± 0.4 C ± ± ± ± ± ± 6.6 * 22 ± 2.4 * 310 ± ± ± A ± ± ± ± ± ± ± 3.5 * 760 ± 36 * 46 ± ± 0.7 B ± ± ± ± ± ± 2.2 * 31 ± 2.3 * 840 ± 23 * 50 ± 1.3 * 21 ± 0.8 C ± ± ± ± ± ± 9.6 * 26 ± 2.7 * 440 ± 67 * 28 ± ± A ± ± ± ± ± ± 4.9 * 80 ± 6.1 * 1710 ± ± ± 3.2 B ± ± ± ± ± ± ± ± ± ± 2.9 C ± ± ± ± ± ± ± 10* 1240 ± ± ± A ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 6.8 B ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 6.3 * 54 ± 12 * 2890 ± ± 25 * 67 ± 8.0 * C ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 17 * 130 ± ± ± ± 5.3 * Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 24/27

25 Note. Values are means ± SE. * Indicates statistically significant (p<0.05) effects of TCDD compared to respective control. Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 25/27

26 Table 3: The effects of gestational day 15 TCDD exposure (1 µg/kg TCDD) on body weight (g) and anogenital distance (mm) in male progeny necropsied at postnatal day (PND) 1 or 4. PND Rat line Note. Values are means ± SE. * Indicates statistically significant (p<0.05) effects of TCDD compared to respective control. Dose (µg/kg TCDD) N Body weight Anogenital distance (g) (mm) 1 A ± ± ± ± 0.1 B ± ± ± ± 0.2 C ± ± ± ± 0.2 * 4 A ± ± ± ± 0.3 B ± ± ± ± 0.3 C ± ± ± ± 0.2 Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 26/27

27 Figures Fig. 1: Effect of in utero and lactational exposure to TCDD on daily sperm production (DSP) and cauda epididymal sperm reserve (mean ± SE) at the age of 70 days in line A, B and C rats (N=4-7 litters /treatment). Means significantly (P < 0.05) different from corresponding controls are depicted with solid symbols. Fig. 1 DSP (10 6 / testis) Sperm (10 6 /CE) Daily sperm production // A B C 100 // Cauda epididymal (CE) sperm reserve // 100 // Maternal TCDD dose (µg/kg) Ulla Simanainen Saved 3/31/2004 2:04 PM, Printed 3/31/2004 6:07 AM 27/27